Today’s research tools and experiments help scientists zoom in ever closer on the mechanics of many kinds of cancer. In the past, clinicians defined most cancers by phenotype, but molecular signatures now identify many cancers. The work on cancer markers also reveals some of the steps that initiate and promote this diverse family of diseases. As a result, some of today’s cancer researchers believe that targeted therapeutics—aiming attacks at defective molecules—is maturing in its development.

Every country needs new techniques for fighting cancer. According to Rafael Bengoa, director of management of chronic disease at the World Health Organization, 15 million to 16 million new cases of cancer emerge every year. Cancer kills millions every year, as well. In 2000, for example, cancer killed 6 million people, making this group of diseases responsible for 12 percent of worldwide deaths that year. Worst of all, Bengoa and his colleagues expect the number of people diagnosed with cancer to keep increasing by 50 percent every year into the foreseeable future. “So far, we’ve just seen the tip of the cancer iceberg,” Bengoa says.

In general, cancerous cells divide continuously. Consequently, the ever-multiplying cells invade tissues and organs. Cancerous cells can even metastasize, essentially jumping from one location to another. Despite all of the research on cancer, no one knows precisely what causes it. Scientists do know, however, that a range of factors—genetic predisposition, exposure to chemicals or viruses, and even stress—could set off this disease. Many scientists also suspect that the clues to the cause of cancer lie in a better understanding of signal transduction and its impact on cell division. Work on the human genome and those of model organisms helps reveal which genes might play a part in cancer. For example, researchers know that mutations of about 300 human genes can unleash division in cells, making them cancerous.

As a result, new tools that compare the activity of these genes in normal and mutated states could point to new ways to stop the division, or kill the cancer. This article explores a wide range of new tools and techniques—from following phosphorylation to knocking down genes with RNA interference—that help scientists develop a combination of therapeutics that could rein in the wildly growing cells.

Deciphering the Signals
“A huge number of intercellular connections make up a network that lets cells sense the environment and respond to stimuli,” says Christopher Bunker, director of new business development at Cell Signaling Technology. Those networks make up pathways that lie at the foundation of signal transduction, or turning environmental stimuli—hormones, DNA damage, cytotoxic substances, and so on—into actions. The signals leading to actions cascade through a collection of protein-protein and protein-substrate interactions. In fact, Bunker says, scientists currently know of 518 kinases from the human genome that participate in signal transduction. He pauses before he concludes: “It is amazingly humbling.” To try to keep up-to-date, see “Signal Transduction Online.”

More knowledge about signal transduction, however, points out places where cancer might start or, later, prosper. Just one mutation could lead to a modified protein that pushes cell division into a runaway process. To study these signal transduction pathways, scientists require a variety of supplies, and companies including EMD Bioscience, ICN Biomedicals, and Sigma-Aldrich provide a broad line of the basic compounds and reagents used in this field. Likewise, some companies—including Alexis Corporation, Biomol, and BD Biosciences Pharmingen—focus largely on reagents and kits made specifically for signal transduction research. Focusing even more closely, Cell Signaling Technology, Santa Cruz, and Upstateconcentrate on one aspect of signal transduction research, such as antibodies.

For example, Bunker says, “Our company is dedicated to signal transduction, with a particular focus on cancer research.” Specifically, scientists at Cell Signaling Technology develop reagents and antibodies that track phosphorylation, which indicates the activity level of kinases. “This tells us if a kinase is ‘on’ or ‘off,’” Bunker says. “In essence, researchers use our reagents to see how cancer changes signal transduction.” For instance, a mutation could change the activity of a variety of kinases.

Bunker also points out that his company’s reagents can be used in high throughput screening to find compounds that inhibit a kinase. “Researchers can even look at clinical samples from an individual who has cancer and study the protein phosphorylation in that specific case, which is just the beginning of personalized medicine,” Bunker says.

Making such detection systems and assays work, though, depends on antibodies that work, as well. “One of our strengths,” says Bunker, “is an expert team that validates antibodies, showing that they are specific and work as advertised for various applications.” In addition, Cell Signaling Technology is now developing rabbit monoclonal antibodies that, Bunker states, “supply high affinity and clonal uniformity.”

Array Update
Given all of the discoveries that show genetic links to some forms of cancer, researchers want to explore how genes work together and what proteins they produce. Rather than looking at genes or proteins one by one, scientists look simultaneously at hundreds to several thousands with microarrays from companies that include Affymetrix andMergen.

Mergen adapted its ExpressChip DNA Microarray system to high throughput gene expression analysis. Jamie Love, vice president of business development and marketing at Mergen, explains that clients can purchase off-the-shelf arrays or contract Mergen to produce arrays specific to their needs. Love says, “Our high throughput ‘hexadec’ system makes 16 identical arrays, each containing up to a thousand oligos. We have an inventory of oligos designed and tested to determine the expression of thousands of rat, mouse, and human genes. Customers can choose the genes that they want and Mergen will deliver the custom arrays in about a month.”

One reason for high throughput is the need to repeat many experiments to get meaningful results. Love explains, “Mergen synthesizes its oligos off-chip in batches large enough to make thousands of arrays. We quality control the batches and then use contact printing in our clean room manufacturing facility, to deposit the oligos onto polymer coated slides.” He adds, “Thanks to these manufacturing methods—and others that are proprietary—Mergen has the highest array-to-array consistency. That lowers the need to repeat experiments, saving time, money, and valuable samples.”

Mergen also offers a full hybridization service. Clients send Mergen their RNA, the company makes the probes, hybridizes it to the arrays, collects the data, and sends them to the client. Love adds, “Using Mergen’s hexadec, one technician needs only two days to hybridize and collect data from 192 experiments.”

Once a cancer researcher knows which genes participate in a specific disease, a protein microarray can pull out the peptides that get created. For example, Eric Fung, Ciphergen’s director of clinical affairs, says, “We sell the ProteinChip Biomarker System, which is a complete system for analyzing proteins.” It includes a ProteinChip array to bind the sample, a ProteinChip reader to analyze the bound sample, and ProteinChip software to analyze the data. Fung recommends this system for basic science—including the study of posttranslational modifications or protein-protein interactions—and clinical studies, such as comparing protein expression in subjects with and without cancer. “Once we have discovered the proteins of interest through protein expression profiling,” Fung says, “we can develop quantitative assays for these markers on the same ProteinChip system that was used to make the discovery.”

Gene Knockout
To determine how a gene works, scientists can turn it off in a whole animal and see what happens, as in a knockout mouse. Now, researchers can purchase knockout mice or have them created on a contract basis by several companies, including Charles River Laboratories, The Jackson Laboratory, and Taconic. Anthony F. Trombino, senior manager of Oncology Research Services at Charles River Laboratories, says, “A knockout mouse is pretty straightforward: take out a gene that you suspect might be involved in cancer and examine the resulting phenotype.” Knocking out either a proto-oncogene or a tumor suppressor gene, for example, could make a mouse more susceptible to developing tumors, potentially allowing tumors to arise more quickly and grow faster.

The Disease Models Program from Charles River Laboratories provides researchers with rodent models that express common human diseases, including diabetes, obesity, and hypertension. This company also offers immunodeficient mice for cancer research. Trombino says these mice let researchers monitor tumor growth over time, and, “You can look at a compound’s efficacy and toxicity using these models.” James A. Jersey, president of Charles River Proteomic Services, a division of Charles River Laboratories, adds: “Knockout mice are primarily used in studies seeking to understand a gene’s role in disease pathways and thus indirectly potential drug targets.”

Charles River Laboratories also provides other tools for cancer research. Paul A. Oskar, III, general manager of discovery and development services at the Worcester, Massachusetts, division, says, “Our laboratories provide services ranging from identification of novel protein biomarkers through high volume targeted biomarker screening of tissue and blood samples.” He adds, “Through an alliance with Rules-Based Medicine, we can very efficiently and sensitively screen panels of potential biomarkers in a targeted fashion for plasma, urine, and other biofluids using very small sample volumes—for example, 10 microliters.” Changes in the concentration patterns of targeted analytes can prove useful in understanding effects of treatments, population phenotyping, and so on. Jersey and his colleagues apply tools in proteomics in the search for identification of potential novel protein-based biomarkers. He says, “Charles River is involved in the full spectrum of cancer research with the exception of the clinic.”

In some cases, a scientist might prefer a cell line over an entire animal. For those situations, ArtisOptimus grows primary mouse embryonic fibroblasts (MEFs) that retain their initial growth and genetic properties. “The idea was to take primary mouse cells harvested from embryos,” says Jason Weber assistant professor at Washington University and founder of ArtisOptimus, “and obtain a low-pass number cell line that is easy to culture, even for a novice, and available for immediate use.” In addition, the cells come directly from a knockout mouse, so this combines a genetic manipulation with a cell line. By growing a large population of identical cells, a researcher can use them to explore signal pathways or test compounds as drugs. “This approach gives researchers a lot more confidence in their cell types and in the genetic makeup of the cell,” says Patrick Dillion president and chief executive officer at ArtisOptimus.

The MEF wild type and knockout cell lines from ArtisOptimus will start to become available by the middle of 2004, says Alison Rowland, vice president of sales and marketing at ArtisOptimus. The first MEFs will aim at various cancer-related gene families, including growth factors, signal transduction factors, tumor suppressors, and so on. “This innovation will bring primary mouse cell technology to the research community when it has previously been outside of their reach due to significant time and cost investment,” says Weber.

Exploiting Interference
During the mid 1990s, researchers discovered RNA interference (RNAi), which can knock down a specific gene. Double stranded RNA sets off a process that cuts up homologous messenger RNA. Consequently, the gene that made that mRNA gets effectively shut down. The field of RNAi soon generated many products from a wide variety of companies, includingDharmacon and Upstate.

At Dharmacon, Stephen Scaringe, chairman and chief scientific officer, says, “We make a small, interfering RNA [siRNA] technology platform.” That platform includes custom services. “With our proprietary synthesis platform,” says Scaringe, “we can make any siRNA.” Dharmacon also offers 1,500 reagents in its siGENE list, so that researchers can look for siRNAs to block specific genes. During the summer of 2003, Dharmacon also introduced its On-Target technology, which aims at increased specificity. “This reduces off-target effects,” says Scaringe.

Once inside a cell, siRNA can prove unstable, and the gene knockdown stops as soon as the siRNA gets disrupted. To battle that problem and provide longer lasting knockdowns, Dharmacon created its siSTABLE, which company literature describes as “a novel proprietary form of siRNA in which the siRNA strands have been chemically modified to dramatically enhance stability and silencing longevity.” According to Scaringe, typical siRNA lasts five days, but the siRNA from siSTABLE lasts seven to 10 days, even two weeks in some cases. “It’s better distributed in the cell, as well,” says Scaringe. While nonspecific interactions from siRNA can down-regulate some genes unintentionally, siRNA can also up-regulate some genes—also inadvertently. “It was unexpected,” says Scaringe, “ but our siSTABLE creates much fewer up-regulated events.”

To make RNAi work, though, the material must get to the right place. Jim Hagstrom, vice president of scientific operations at Mirus, says, “The main way to get siRNA into specific cells is through transfection.” He adds, “Transfection has been around a long time, and we’ve been transfecting genes and plasmid DNA into cells for some time.”

It turned out, though, that transfection worked more easily with siRNA than with genes. The genes, for one thing, must get in the nucleus to do their jobs. On the other hand, siRNA does its work in the cytoplasm. In addition, the siRNA is smaller. So it moves around more easily. To make transfection work, though, Mirus needed the right reagents. “We started looking at our previously developed transfection agents for genes,” says Hagstrom, “but some of them didn’t work ideally. So we worked hard at formulating the best reagents for delivering siRNA to cells in culture.” Mirus ended up with a mixture of a polymer and an amphipathic polyamine that works for many cell types and many siRNAs. Still, some cells resist transfection, but it is less of a problem with siRNA than with genes, says Hagstrom. The culmination of testing reagents at Mirus produced its TransIT-TKO product.

RNA interference provides lots of angles for cancer research. Hagstrom says that siRNAs can be used to knockdown expression of specific genes in a wide range of cancerous cell lines. The expression profile obtained using siRNA knockdown can be particularly useful for understanding gene relationships in the disease process and identifying or confirming new therapeutically relevant target genes. Hagstrom says, “It’s a very powerful tool.”

A Future of Fighting
Ongoing projects aim a variety of agents at cancer. A handful of pharmaceutical advances, for example, come from Novartis. This company explores angiogenesis inhibition, apoptosis, hormone therapy, metastasis, microtubule stabilization, signal transduction, and other areas in search of drug targets and potential compounds. As a result, Novartis produced several drugs, including Glivec, which attacks chronic myelogenic leukemia. In discussing Glivec, Alexander Kamb, vice president and global head of oncology research at Novartis Institutes for Biomedical Research, says, “This drug arguably represents a revolution comparable to the first chemotherapeutics discovered in the 1950s.” He says that Glivec causes few side effects—none in some patients—and can turn some cancers into chronic rather than acute diseases. Kamb adds, “We also have an antiangiogenesis compound for colon cancer in Phase III trials. This is an exciting possibility because an angiogenesis inhibitor is potentially broadly applicable. All tumors need blood.” For more treatment updates, see "New Waves of Therapeutic Manufacturing."

As researchers continue the search for new tools against cancer, Rafael Bengoa of the World Health Organization already knows his favorite. “There is already an intervention,” he says, “that can prevent a hundred thousand new cases of cancer in the U.S. and stop 60 thousand deaths, every year.” Prevention is Bengoa’s powerful intervention. “We don’t need to wait,” he says. “We don’t need a flashy new discovery.” Instead, he encourages countries to organize public health interventions for prevention and early diagnosis.

Bengoa recommends fighting cancer at every opportunity. Consequently, he advocates a combination of prevention and therapy. “There’s an obvious molecular basis in all cancer formations,” he says. “It’s obvious that we should expect some quite spectacular advances.” The environment and genes combine to create cancer, so clinicians, government officials, basic researchers, and public health professionals must simultaneously fight the causes—tobacco, other carcinogens, wayward genes, and who knows what—and symptoms. In the end, only a multipronged attack stands a chance against a family of diseases as diverse as cancer.